US20070179423A1 - Adjusting ph in a method of separating whole blood - Google Patents

Abstract

This invention is directed to a method of collecting and separating whole blood into components. The method includes the steps of adding an anticoagulant having an acidic pH to a bag for collecting and/or separating whole blood, collecting whole blood in the bag, loading the bag containing anticoagulated whole blood on a rotor, spinning the bag on the rotor to separate the whole blood into at least one component; and squeezing the bag on the rotor to push the component from the separation bag into at least one satellite bag.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
• This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 60/766,586 filed Jan. 30, 2006.
BACKGROUND
• For transfusions of blood and blood components, whole blood is typically separated into three components: plasma, red blood cells and platelets.
• There are traditionally two ways to obtain these blood components. One way is to collect whole blood from donors/patients and separate it into components manually at some time period after the whole blood collection. Using this method, whole blood is collected into FDA-approved containers that are pyrogen-free and sterile, and contain sufficient anticoagulant for the quantity of blood to be collected. Whole blood which is collected in this way is separated into components manually in a lab by a technician, and separation typically occurs from between about 2 and 8 hours after collection in the United States, and between about 2 to 24 hours in Europe.
• Another way to separate whole blood into components is by using apheresis or automated cell-separation devices. Apheresis devices separate whole blood into components automatically, and return any uncollected blood components back to the donor during the collection procedure.
• An alternative to manual processing of whole blood as described above is the automatic processing of previously collected whole blood using an automated whole blood processing device such as the Atreus machine, manufactured by Gambro BCT, Inc. (Lakewood, Colo., USA.)
• In whole blood processing, (whether by hand or by an automated machine), and in apheresis the addition of anticoagulant to the blood is necessary to prevent the formation of blood clots. In manual whole blood processing, blood is collected from a donor/patient directly into a bag that contains an approved anticoagulant-preservative solution designed to both prevent clotting and maintain cell viability and function during storage. In manual whole blood processing, whole blood is collected in CPD (citrate-phosphate-dextrose) anticoagulant.
• In apheresis processing, the anticoagulant ACDA (acid-citrate-dextrose formula A) is added to the blood withdrawn from a donor/patient at the beginning of the collection procedure.
• It is to the optimal collection of platelets and to the optimal leukoreduction of red blood cells from whole blood processed on an automated blood processing device that the present invention is directed.
BRIEF SUMMARY OF THE INVENTION
• This invention is directed to a method of collecting and separating whole blood into components. The method includes the steps of adding an anticoagulant having an acidic pH to a bag for collecting and/or separating whole blood, collecting whole blood in the bag, loading the bag containing anticoagulated whole blood on a rotor, spinning the bag on the rotor to separate the whole blood into desired components; and squeezing the bag on the rotor to push the desired components from the separation bag into satellite bags.
• This invention also includes a method of leukoreducing red blood cells separated from previously collected and stored whole blood. The steps include collecting whole blood in CPD anticoagulant, storing the anticoagulated whole blood overnight, loading the anticoagulated whole blood on a rotor, spinning the rotor to separate the stored whole blood into at least a red blood cell component, and squeezing the blood on the rotor to push at least the red blood cells component into a satellite bag, increasing the pH of the separated red blood cell component in the satellite bag, and leukoreducing the red blood cell component.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 is a schematic view of a set of separation and collection bags designed for cooperating with an automated whole blood separation apparatus.
• FIG. 2 is a schematic view, partly in cross-section, of a whole blood separation apparatus which may be used with the present invention.
• FIG. 3 is a schematic view, partly in cross-section, of a whole blood separation apparatus which may be used with the present invention.
• FIG. 4 is a schematic view of another set of separation and collection bags designed for cooperating with another automated whole blood separation apparatus.
• FIG. 5 is a schematic view, partly in cross-section, of a whole blood separation apparatus which may be used with the present invention.
• FIG. 6 is a top view of the rotor of the separation apparatus ofFIG. 5.
• FIG. 7 in schematic view, in cross-section along a radial plane, of a separation cell of the separation apparatus ofFIGS. 5-7.
DETAILED DESCRIPTION
• This invention is for use with automated blood separation devices for separating collected whole blood into components. The whole blood may be separated into components immediately after collection from a donor, or may be separated into components from whole blood which was previously collected from a donor. Previously collected means that the whole blood was collected from a donor at some period of time prior to the blood being separated in the automated blood separation device. The device described below is described in patent application PCT/US2006/031492, herein incorporated by reference in its entirety to the amount not inconsistent.
• FIG. 1 shows an example of a set of bags adapted to the separation of whole blood into a plasma component essentially comprising plasma, a first blood cell component essentially comprising mononuclear cells and platelets, and a second blood cell component essentially comprising red blood cells. This bag set comprises a flexible separation bag1 and threeflexible product bags2,3,4 connected thereto. The separation bag1 comprises anannular separation chamber5 having a substantially circular outer edge6 and an innercircular edge7. The outer circular edge6 and the innercircular edge7 of theseparation chamber5 are substantially concentric. The separation bag1 further comprises a semi-flexible disk-shaped connecting element9 that is connected to theinner edge7 of theannular chamber5. The disk-shaped connecting element9 comprises adistribution channel10 embedded therein, which communicates through apassage11 with theannular chamber5. Thedistribution channel10 substantially extends along an arc of circle. The disk-shaped connecting element9 comprises a series ofholes12 for securing the separation bag1 to the rotor of a centrifuge.
• Thefirst satellite bag2 has two purposes and is successively used as both a blood collection bag and as a mononuclear cell/platelet component bag. The first satellite bag is intended for initially receiving a volume of whole blood from a donor (usually about 450 ml) before the separation process, and the mononuclear cell/platelet component during the separation process. Thefirst satellite bag2 is flat, substantially rectangular, and comprises two reinforced ears at its uppercorners having holes13 for hanging the bag. It is connected to the separation bag1 by afirst transfer tube14, fitted with aclamp15. Thefirst transfer tube14 has a first end connected to the upper edge of thefirst satellite bag2 and a second end connected to a first end of thedistribution channel10.
• Anticoagulant is added to thefirst satellite bag2. Typically about 63 ml of anticoagulant solution is added to a blood donation of about 450 ml. The anticoagulant may be added to thefirst satellite bag2 before the blood is added, or may be added after the blood is added. Aplug16 removable from within the first satellite bag2 (so-called “frangible pin”, for example) blocks a liquid flow through thefirst transfer tube14 and prevents the anticoagulant solution from flowing from thefirst satellite bag2 into the separation bag1.
• Acollection tube17 is connected at one end to the upper edge of thefirst satellite bag2 and comprises, at the other end, a needle protected by asheath18. Afrangible pin19 removable from within thefirst satellite bag2 plugs the downstream end of thecollection tube17 and prevents the anticoagulant solution from flowing out of thefirst satellite bag2 through thecollection tube17.
• Thesecond satellite bag3 is intended for receiving a plasma component. It is flat, substantially rectangular, and comprises two reinforced ears at its uppercorners having holes13 for hanging the bag. It is connected by asecond transfer tube20 to the separation bag1. Thesecond transfer tube20, which is fitted with aclamp15, has a first end connected to the upper edge of thesecond satellite bag3 and a second end connected to a second end of thedistribution channel10.
• Thethird satellite bag4 is intended for receiving a red blood cell component. It is flat, substantially rectangular, and comprises two reinforced ears at its uppercorners having holes13 for hanging the bag. It is connected by athird transfer tube21 to the separation bag1. Thethird transfer tube21 has a first end connected to the upper edge of thethird satellite bag4 and a second end that is connected to thedistribution channel10 so as to face thepassage11 between thedistribution channel10 and theseparation chamber5. It comprises two segments respectively connected to the inlet and the outlet of a leuko-reduction filter22. The tube segment connected to the separation bag1 is fitted with aclamp15. Thefilter22 may be, for example, a filter of the type RC2D manufactured by Pall Corporation. Such a filter comprises a disk-shaped casing to which radial inlet and outlet ports are connected, in diametric opposition. Thethird satellite bag4 contains a volume of storage solution for red blood cells. The storage solution may be added to thethird satellite bag4 either before the cells are added or after the cells are added. Aplug23 removable from within the third satellite bag4 (so-called “frangible pin”, for example) blocks a liquid flow through thethird transfer tube21 and prevents the storage solution from flowing from thethird satellite bag4 into the separation bag1.
• FIGS. 2 and 3 show an embodiment of an apparatus for separating a volume of composite liquid by centrifugation. The apparatus comprises a centrifuge adapted for receiving the separation bags shown in figures, and a component transferring means for causing the transfer of separated components into the satellite bags.
• The centrifuge comprises a rotor that is supported by a bearingassembly30 allowing the rotor to rotate about a verticalcentral axis31. The rotor comprises:
• acylindrical rotor shaft32,33;
• acentral compartment34 for containing satellite bags, which is connected to therotor shaft32,33 at the upper end thereof;
• a support member87 (not shown inFIGS. 3 and 4) for supporting at least one satellite bag in a determined position within thecentral compartment34; and
• acircular turntable35 for supporting a separation bag, which is connected to thecompartment34 at the upper end thereof, the central axes of therotor shaft31,32, thecompartment34 and theturntable35 coinciding with therotation axis31.
• The rotor shaft comprises a firstupper portion32 and a secondlower portion33. Theupper portion32 of the shaft extends in part through the bearingassembly30. Apulley36 is connected to the lower end of theupper portion32 of the shaft.
• The centrifuge further comprises amotor40 coupled to the rotor by abelt41 engaged in a groove of thepulley36 so as to rotate the rotor about the centralvertical axis31.
• The separation apparatus further comprises a first, second and thirdpinch valve members42,43,44 (seeFIG. 1) that are mounted on the rotor for selectively blocking or allowing a flow of liquid through a flexible plastic tube, and selectively sealing and cutting a plastic tube. Eachpinch valve member42,43,44 comprises an elongated cylindrical body and a head having a groove that is defined by a stationary upper jaw and a lower jaw movable between an open and a closed position, the groove being dimensioned so that one of thetransfer tubes14,20,21 of the bag set shown inFIG. 1 can be snuggly engaged therein when the lower jaw is in the open position. The elongated body contains a mechanism for moving the lower jaw and it is connected to a radio frequency generator that supplies the energy necessary for sealing and cutting a plastic tube. Thepinch valve members42,43,44 are mounted at the periphery of thecentral compartment34 so that their longitudinal axes are parallel to thecentral axis31 of the rotor and their heads protrude above the rim of thecompartment34. The position of thepinch valve members42,43,44 with respect to the separation bag1 and thetransfer tubes14,20 connected thereto when the separation bag1 is mounted on theturntable35 is shown in dotted lines inFIG. 1. Electric power is supplied to thepinch valve members42,43,44 through aslip ring array45 that is mounted around thelower portion33 of the rotor shaft.
• Theturntable35 comprises a central frusto-conical portion46, the upper, smaller edge of which is connected to the rim of thecompartment34, an annularflat portion47 connected to the lower, larger edge of the frusto-conical portion46, and an outercylindrical flange48 extending upwards from the outer periphery of theannular portion47. Theturntable35 further comprises a vaultedcircular lid49 that is secured to theflange48 by a hinge so as to pivot between an open and a closed position. Thelid49 is fitted with alock51 by which it can be blocked in the closed position. Thelid49 comprises a large cut-out in its upper part that gives access to thecentral compartment34 of the rotor. Thelid49 has an annular interior surface that is so shaped that, when thelid49 is in the closed position, it defines with the frusto-conical portion46 and the annularflat portion47 of the turntable38 a frusto-conical annular compartment53 having a radial cross-section that has substantially the shape of a parallelogram. The frusto-conical annular compartment53, later the “separation compartment”, is intended for containing the separation bag1.
• The component transferring means comprises a squeezing system for squeezing the separation bag within the separation compartment53 and causing the transfer of separated components into the satellite bags. The squeezing system comprises a flexibleannular diaphragm54 that is so shaped as to line the frusto-conical portion46 and the annularflat portion47 of theturntable35, to which it is secured along its smaller and larger circular edges. The squeezing system further comprises ahydraulic pumping station60 for pumping a hydraulic liquid in and out an expandablehydraulic chamber55 defined between theflexible diaphragm54 and theturntable35, via aduct37 extending through the rotor from the lower end of thelower portion33 of the rotor shaft to theturntable35. The pumpingstation60 comprises a piston pump having apiston61 movable in a hydraulic cylinder62 fluidly connected via arotary fluid coupling38 to therotor duct37. Thepiston61 is actuated by a stepper motor63 that moves alead screw64 linked to the piston rod. The hydraulic cylinder62 is also connected to a hydraulicliquid reservoir65 having an access controlled by avalve66 for selectively allowing the introduction or the withdrawal of hydraulic liquid into and from a hydraulic circuit including the hydraulic cylinder62, therotor duct37 and the expandablehydraulic chamber55. Apressure gauge67 is connected to the hydraulic circuit for measuring the hydraulic pressure therein.
• The separation apparatus further comprises threesensors56,57,58 for detecting characteristics of the separation process occurring within a separation bag when the apparatus operates. The threesensors56,57,58 are embedded in thelid49 at different distances from the rotation axis of the rotor, afirst sensor56 being the farthest to the rotation axis, athird sensor58 being the closest to the rotation axis and asecond sensor57 occupying an intermediate position. When thelid49 is closed, the threesensors56,57,58 face the separation bag1 as shown inFIG. 1. The first sensor56 (later the “bag sensor”) is embedded in thelid49 so as to be positioned over theseparation chamber5, at about one third of the width of the separation chamber from the inner edge6 thereof, and it is offset with respect to thepassage11 between theseparation chamber5 and thedistribution channel10. Thebag sensor56 is able to detect the presence or absence of a liquid in theseparation chamber5, as well as red blood cells in a liquid. The second sensor57 (later the “bay sensor”) is embedded in thelid49 so as to be positioned over thepassage11 between theseparation chamber5 and thedistribution channel10. Thebay sensor57 is in the pathway of any component flowing from theseparation chamber5 into the threesatellite bags2,3,4. Thebay sensor57 is able to detect the presence or absence of a liquid in thedistribution channel10 as well as to detect red blood cells in a liquid. The third sensor58 (later the “channel sensor”) is embedded in thelid49 so as to be positioned over thedistribution channel10. Thechannel sensor58 is in the pathway of any component flowing from theseparation chamber5 into thesecond satellite bag3. Thechannel sensor58 is able to detect the presence or absence of a liquid in thedistribution channel10 as well as to detect red blood cells in a liquid. Eachsensor56,57,58 can comprise a photocell including an infra-red LED and a photo-detector. Electric power is supplied to thesensors56,57,58 through theslip ring array45.
• The separation apparatus further comprises acontroller70 including a control unit (microprocessor) and a memory for providing the microprocessor with information and programmed instructions relative to various separation protocols and to the operation of the apparatus in accordance with such separation protocols. In particular, the microprocessor is programmed for receiving information relative to the centrifugation speed(s) at which the rotor is to be rotated during the various stages of a separation process, and information relative to the various transfer flow rates at which separated components are to be transferred from the separation bag1 into thesatellite bags2,3,4. The information relative to the various transfer flow rates can be expressed, for example, as hydraulic liquid flow rates in the hydraulic circuit, or as rotation speeds of the stepper motor63 of thehydraulic pumping station60. The microprocessor is further programmed for receiving, directly or through the memory, information from thepressure gauge67 and from thephotocells56,57,58 and for controlling thecentrifuge motor40, the stepper motor63, and thepinch valve members42,43,44 so as to cause the separation apparatus to operate along a selected separation protocol.
• An example of a first separation protocol aimed at the preparation of three blood components, namely a plasma component, a first blood cell component essentially comprising platelets, and a second blood cell component essentially comprising red blood cells, is explained below. This first separation protocol does not require the use of thechannel sensor58. The operation of the separation apparatus along the first separation protocol is as follows:
• In the first separation stage, a bag set as shown inFIG. 1, in which a satellite bag contains a volume of whole blood, is set in place in the rotor of a centrifuge (as shown inFIGS. 2,3).
• At the onset of the first stage, thefirst satellite bag2 of the bag set ofFIG. 1 contains a volume of anticoagulated whole blood (usually about 500 ml). Thecollection tube17 has been sealed and cut. The clamps15 on thetransfer tubes14,20,21 connecting thesatellite bags2,3,4 to the separation bag1 are closed. Thefrangible pin16 blocking communication between thefirst satellite bag2 and the separation bag1 is broken as well as thefrangible pin23 blocking communication between thethird satellite bag4 and the separation bag1. Thefirst satellite bag2 and thethird satellite bags4 are engaged on the first couple of pegs of a bag holder (not shown), thefirst satellite bag2 being engaged first. Thesecond satellite bag3 is engaged on the second couple of pegs (not shown). The bag holder is mounted in a cradle (not shown), as a result of which thefirst satellite bag2 is adjacent to the inner surface of the cradle. The cradle is then fully inserted into thecentral compartment34 of the centrifuge. Thesatellite bags2,3,4 are then substantially located on one side of a plane containing the rotation axis of therotor31. The collection bag1 is laid on theturntable35 and the pins on the flange of the rotor liner are engaged in theholes12 of the disk-shaped connecting element9 of the separation bag1. Thefirst transfer tube14 connecting thefirst satellite bag2 to the separation bag1 is engaged in the firstpinch valve member42, thesecond transfer tube20 connecting thesecond satellite bag3 to the separation bag1 is engaged in the thirdpinch valve member44, and thethird transfer tube21 connecting thethird satellite bag4 to the separation bag1 is engaged in the secondpinch valve member43. The clamps15 on thetransfer tubes14,20,21 connecting thesatellite bags2,3,4 to the separation bag1 are opened. Thelid49 of the rotor is closed.
• In the second stage, the anticoagulated whole blood contained in thefirst satellite bag2 is transferred into the separation bag1.
• At the onset of the second stage, the firstpinch valve member42 is open and the second and thirdpinch valve members43,44 are closed. The rotor is set in motion by thecentrifuge motor40 and its rotation speed increases steadily until it reaches a first centrifugation speed (e.g. about 1500 RPM) that is so selected as:
• To be high enough to cause the transfer, under centrifugation forces, of the content of thefirst satellite bag2 into the separation bag1;
• To be high enough to cause the whole transfer to happen in the shorter period of time; while, at the same time,
• To be low enough not to cause pressure within thefirst satellite bag2 to substantially exceed a determined pressure threshold above which hemolysis would occur;
• To be low enough not to generate shearing forces in the flow of blood entering the separation bag1 that would cause hemolysis.
• It has been determined that the pressure threshold above which hemolysis occurs in thesatellite bag2 is about 10 PSI, and that the maximum rotation speed at which such pressure threshold is not reached and the shearing forces in the blood flow entering the separation bag do not cause hemolysis is about 1800 RPM. At a rotation speed of about 1500 RPM, it takes about one minute for transferring about 500 ml of anticoagulated blood from thesatellite bag2 into the separation bag1.
• If thebag cell56 has not detected red blood cell within a predetermined period of time following the start of the centrifugation process, thecontrol unit70 causes the rotor to stop and an alarm to be emitted. This could happen in particular if thefrangible pin16 has not been broken or if theclamp15 on thefirst transfer tube14 has not been opened.
• In the third stage, the blood within the separation chamber is sedimented to a desired level.
• At the onset of this stage, thepinch valve members42,43,44 are closed. The rotor is rotated at a second, high centrifugation speed (for example, about 3200 RPM) for a predetermined period of time (for example, about 220 seconds) that is selected so that, whatever the hematocrit of the whole blood initially transferred in the separation bag1, the blood sediments therein at the end of the predetermined period to a point where the hematocrit of the outer annular red blood cell layer is about 90 and the inner annular plasma layer is substantially devoid of cells. In more details, at the outcome of this sedimentation stage, the separation bag1 exhibits four layers: a first inner layer mainly comprising plasma, a second intermediate layer mainly comprising platelets, a third intermediate layer mainly comprising mononuclear cells (lymphocytes and monocytes), and a fourth outer layer mainly comprising red blood cells (granulocytes remain embedded in the most inner layer of red blood cells).
• In the forth stage, a plasma component is transferred into thefirst satellite bag2.
• At the onset of this stage, thepinch valve members42,43,44 are closed. The rotor is rotated at the same high centrifugation speed as in the sedimentation stage. After a predetermined period of time after thebag sensor56 has stopped detecting red blood cells, which can happen before the end of the predetermined sedimentation period, the thirdpinch valve member44 controlling the access to thesecond satellite bag3 is opened and the pumpingstation60 is actuated so as to pump hydraulic liquid at a constant flow rate (for example, about 220 ml/min) into thehydraulic chamber55. The expandinghydraulic chamber55 squeezes the separation bag1 and causes the transfer of plasma into thesecond satellite bag3. The pumpingstation60 is stopped and the thirdpinch valve member44 is closed after a predetermined period of time has elapsed following the detection of red blood cells by thebay sensor57. A small volume of plasma (for example, about 5 ml) remains in the separation bag1.
• The transfer flow rate of the plasma component (which is directly related to the flow rate of the hydraulic fluid) is selected to be as high as possible without disturbing the platelet layer so as to avoid contaminating the plasma component with platelets.
• In the fifth stage a platelet/mononuclear cell component is transferred into thefirst satellite bag2.
• The fifth stage can start as soon as the thirdpinch valve member44 is closed at the end of the fourth stage. At the onset of this fifth stage, thepinch valve members42,43,44 are closed. The rotor is rotated at the same high centrifugation speed as previously. The firstpinch valve member42 controlling the access to thefirst satellite bag2 is opened and the pumpingstation60 is actuated so as to pump hydraulic liquid at a constant flow rate (for example, about 140 ml/min) into thehydraulic chamber55. The expandinghydraulic chamber55 squeezes the separation bag1 and causes the transfer, into thefirst satellite bag2, of a platelet/mononuclear cell component comprising the residual volume of plasma, the platelets, lymphocytes, monocytes and a small amount of red blood cells. The pumpingstation60 is stopped and the firstpinch valve member42 is closed after a predetermined volume has been transferred into thefirst satellite bag2, that is also after a predetermined amount of time has elapsed for a given hydraulic liquid flow rate. This predetermined volume of platelet/mononuclear cell component depends in part on the residual amount of plasma in the separation bag1 at the end of the fourth stage. For example, when the residual volume of plasma in the separation bag1 is determined by thebay sensor57, the predetermined volume of the platelet/mononuclear cell component can be set at about between 10 and 15 ml, including about 5 ml of plasma and about 5 ml of red bloods cells.
• In the sixth stage the storage solution for red blood cells contained in thethird satellite bag4 is transferred into the separation bag1.
• The sixth stage can start as soon as the thirdpinch valve member42 is closed at the end of the fifth stage. At the onset of this fifth stage, thepinch valve members42,43,44 are closed. The rotor is rotated at the same high centrifugation speed as previously. The secondpinch valve member43 controlling the access to thethird satellite bag4 is opened, allowing the storage solution contained in thethird satellite bag4 to flow, under centrifugation forces, from thethird satellite bag4 into the separation bag1, through thefilter22. After a predetermined period of time has elapsed after the opening of the secondpinch valve member43, the rotor is sharply braked so that its rotation speed decreases rapidly to a third, reduced speed (for example, 1500 RPM), so as to cause a suspension of the red blood cells contained in the separation bag in the storage solution and lower the viscosity thereof.
• In the seventh stage a red blood cell component is transferred into thethird satellite bag4.
• The seventh stage can start after a predetermined period of time has elapsed after the rotor rotates at the third rotation speed. At the onset of this stage the secondpinch valve member43 controlling the access to thethird satellite bag4 is open and thepinch valve members42,44 are closed. The rotor rotates at the third rotation speed. The pumpingstation60 is actuated so as to pump hydraulic liquid at a first flow rate into thehydraulic chamber55 and consequently squeeze the separation bag1 so as to cause the transfer, through thefilter22, of a red blood cell component into thethird satellite bag4. The first transfer flow rate of the red blood cell component (which is directly related to the flow rate of the hydraulic fluid) is selected to be as high as possible without damaging the red blood cells (hemolysis). When the pressure of the hydraulic liquid measured by thepressure gauge67 reaches a first high pressure threshold, the flow rate of the hydraulic liquid is decreased from the first flow rate to a second flow rate. When the pressure of the hydraulic liquid measured by thepressure gauge67 reaches a second high pressure threshold, the flow rate of the hydraulic liquid is further decreased from the second flow rate to a third flow rate. The second and third transfer flow rates of the red blood cell component are selected so that a maximal portion of the red blood cell component is transferred into thethird satellite bag4. The white blood cells (granulocytes and residual monocytes and lymphocytes) are trapped by thefilter22, so that the ultimate packed red blood cell component in thethird satellite bag4 is substantially devoid of white blood cells.
• In the eighth stage the centrifugation process is ended.
• When a predetermined period of time (for example, about 30 seconds) has elapsed after the pressure of the hydraulic liquid has reached the second pressure threshold, the rotation speed of the rotor is decreased until the rotor stops, the pumpingstation60 is actuated so as to pump the hydraulic liquid from thehydraulic chamber55 at a high flow rate (for example, about 800 ml/min) until it thehydraulic chamber55 is empty, and the threepinch valve members42,43,44 are actuated so as to seal and cut thetubes14,20,21.
• Another automatic whole blood processing system with which the present invention can be used is shown inFIGS. 4 through 7. This automatic whole blood processing system is described in patent application PCT/US2006/21827, herein incorporated by reference in its entirety to the amount not inconsistent.
• FIG. 4 shows an example of a set of bags adapted to the separation of a composite liquid (e.g. whole blood) into a first component (e.g. a plasma component), an intermediate component (e.g. a platelet component), and a second component (e.g. a red blood cell component). This bag set comprises aflexible separation bag1000 and threeflexible satellite bags200,300,150 connected thereto.
• When the composite liquid is whole blood, theseparation bag1000 has two purposes, and is successively used as a collection bag and as a separation bag. It is intended for initially receiving a discrete volume of whole blood from a donor (usually about 450 ml) and to be used later as a separation chamber in a separation apparatus. Theseparation bag1000 is flat and generally rectangular. It is made of two rectangular sheets of plastic material that are welded together so as to define therebetween an interior space having a main rectangular portion connected to a triangular top downstream portion. Afirst tube400 is connected to the tip of the triangular portion, and a second and athird tubes500,600 are connected to either lateral edges of the triangular portion, respectively. The proximal ends of the threetubes400,500,600 are embedded between the two sheets of plastic material so as to be parallel. Theseparation bag1000 further comprises ahole800 in each of its corners that are adjacent to the threetubes400,500,600. Theholes800 are used to secure the separation bag to a separation cell, as will be described later.
• A volume of anticoagulant (typically about 63 ml for a blood donation of about 450 ml) is initially added to the separation bag, and the first andthird tubes400,600 are fitted at their proximal end with abreakable stopper90,100 respectively, blocking a liquid flow therethrough.
• Thesecond tube500 is a collection tube having aneedle120 connected to its distal end. At the beginning of a blood donation, theneedle120 is inserted in the vein of a donor and blood flows into the collection (separation)bag1000. After a desired volume of blood has been collected in the collection (separation)bag1000, thecollection tube500 is sealed and cut.
• Thefirst satellite bag200 is intended for receiving a plasma component. It is flat and substantially rectangular. It is connected to the distal end of thefirst tube400.
• Thesecond satellite bag300 is intended for receiving a red blood cell component. It is flat and substantially rectangular. It is connected to the distal end of thethird tube600. Thethird tube600 comprises two segments respectively connected to the inlet and the outlet of aleukoreduction filter130. Thesecond satellite bag300 contains a volume of storage solution for red blood cells, and thethird tube600 is fitted at its distal end with abreakable stopper140 blocking a liquid flow therethrough.
• Thethird satellite bag150 is intended to receive a platelet component. Like the first andsecond satellite bags200,300, thethird satellite bag150 is flat and substantially rectangular.
• The bag set also contains a T-shaped three-way connector160 having its leg connected by thefirst tube400 to theseparation bag1000, a first arm connected by afourth tube170 to the first satellite bag200 (plasma component bag), and a second arm connected by afifth tube180 to the third satellite bag150 (platelet component bag).
• FIGS. 5,6,7 show a first embodiment of an apparatus for simultaneously separating by centrifugation four discrete volumes of a composite liquid. The apparatus comprises:
• a centrifuge adapted to receive four bag sets shown inFIG. 4, with the four discrete volumes of a composite liquid contained in the four separation bags;
• a component transferring means for transferring at least one separated component from each separation bag into a satellite bag connected thereto;
• a first balancing means for initially balancing the rotor when the weights of the four separation bags are different; and
• a second balancing means for balancing the rotor when the weights of the separated components transferred into the satellite bags cause an unbalance of the rotor.
• The centrifuge comprises a rotor that is supported by abearing assembly3000 allowing the rotor to rotate around arotation axis310. The rotor comprises:
• acylindrical rotor shaft320 to which apulley330 is connected;
• a storage means comprising a centralcylindrical container340 for containing satellite bags, which is connected to therotor shaft320 at the upper end thereof so that the longitudinal axis of therotor shaft320 and the longitudinal axis of thecontainer340 coincide with therotation axis310, and
• a frusto-conical turntable350 connected to the upper part of thecentral container340 so that its central axis coincides with therotation axis310. The frusto-conical turntable350 flares underneath the opening of thecontainer340. Fouridentical separation cells4000 are mounted on theturntable350 so as to form a symmetrical arrangement with respect to therotation axis310.
• The centrifuge further comprises amotor360 coupled to the rotor by abelt370 engaged in a groove of thepulley330 so as to rotate the rotor about therotation axis310.
• Eachseparation cell4000 comprises acontainer410 having the general shape of a rectangular parallelepiped. Theseparation cells4000 are mounted on theturntable350 so that their respective medianlongitudinal axes420 intersect therotation axis310, so that they are located substantially at the same distance from therotation axis310, and so that the angles between their medianlongitudinal axes420 are substantially the same (i.e. 90 degrees). The exact position of theseparation cells4000 on theturntable350 is adjusted so that the weight on the turntable is equally distributed when theseparation cells4000 are empty, i.e. so that the rotor is balanced. It results from the arrangement of the separatingcells4000 on theturntable350 that the separatingcells4000 are inclined with respect to therotation axis310 of an acute angle equal to the angle of the frustum of a cone that geometrically defines theturntable350.
• Eachcontainer410 comprises acavity430 that is so shaped and dimensioned as to loosely accommodate aseparation bag1000 full of liquid, of the type shown inFIG. 4. The cavity430 (which will be referred to later also as the “separation compartment”) is defined by a bottom wall, that is the farthest to therotation axis310, a lower wall that is the closest to theturntable350, an upper wall opposite to the lower wall, and two lateral walls. Thecavity430 comprises a main part, extending from the bottom wall, which has substantially the shape of a rectangular parallelepiped with rounded angles, and an upper part, which has substantially the shape of a prism having convergent triangular bases. In other words, the upper part of thecavity430 is defined by two couples of opposite walls converging towards the centralmedian axis420 of thecavity430.
• One interest of this design is to cause a radial dilatation of the thin layer of a minor component of a composite fluid (e.g. the platelets in whole blood) after separation by centrifugation, and makes it more easily detectable in the upper part of a separation bag. As shown inFIG. 5, the two couples of opposite walls of the upper part of theseparation cell4000 converge towards three cylindricalparallel channels440,450,460, opening at the top of thecontainer410, and in which, when aseparation bag1000 is set in thecontainer410, the threetubes400,500,600 extend.
• Thecontainer410 also comprises a hinged lateral lid470 (seeFIG. 7), which is comprised of an upper portion of the external wall of thecontainer410, i.e. the wall that is opposite to theturntable350. Thelid470 is so dimensioned as to allow, when open, an easy loading of aseparation bag1000 full of liquid into theseparation cell4000. Thecontainer410 comprises a fast locking means (not shown) by which thelid470 can be locked to the remaining part of thecontainer410.
• Thecontainer410 also comprises a securing means for securing aseparation bag1000 within theseparation cell4000. The bag securing means comprises twopins480 protruding on the internal surface of thelid470, close to the top ofseparation cell4000, and twocorresponding recesses490 in the upper part of thecontainer410. The twopins480 are so spaced apart and dimensioned as to fit into the twoholes800 in the upper corner of aseparation bag1000.
• The separation apparatus further comprises a component transferring means for transferring at least one separated component from each separation bag into a satellite bag connected thereto. The component transferring means comprises a squeezing system for squeezing theseparation bags1000 within the separation compartments430 and causing the transfer of separated components intosatellite bags200,300,150.
• The squeezing system comprises aflexible diaphragm500 that is secured to eachcontainer410 so as to define anexpandable chamber510 in the cavity thereof. More specifically, thediaphragm500 is dimensioned so as to line the bottom wall of thecavity430 and a large portion of the lower wall of thecavity430, which is the closest to theturntable350.
• The squeezing system further comprises a peripheralcircular manifold520 that forms a ring within theturntable350 extending close to the periphery of theturntable350. Eachexpansion chamber510 is connected to the manifold520 by asupply channel530 that extends through the wall of therespective container410, close to the bottom thereof.
• The squeezing system further comprises ahydraulic pumping station6000 for pumping a hydraulic liquid in and out theexpandable chambers510 within theseparation cells4000. The hydraulic liquid is selected so as to have a density slightly higher than the density of the more dense of the components in the composite liquid to be separated (e.g. the red blood cells, when the composite liquid is blood). As a result, during centrifugation, the hydraulic liquid within theexpandable chambers510, whatever the volume thereof, will generally remain in the most external part of theseparation cells4000. Thepumping station6000 is connected to theexpandable chambers510, through arotary seal690, by aduct560 that extends through therotor shaft320, the bottom and lateral wall of thecentral container340, and, from the rim of thecentral container340, radially through theturntable350 where it connects to themanifold520.
• As shown inFIG. 5, thepumping station6000 comprises a piston pump having apiston610 movable in ahydraulic cylinder620 fluidly connected via a rotary fluid coupling to therotor duct540. Thepiston610 is actuated by astepper motor640 that moves alead screw650 linked to the piston rod. Thehydraulic cylinder620 is also connected to a hydraulic liquid reservoir660 having an access controlled by avalve670 for selectively allowing the introduction or the withdrawal of hydraulic liquid into and from a hydraulic circuit including thehydraulic cylinder620, therotor duct560 and the expandablehydraulic chambers510. Apressure gauge680 is connected to the hydraulic circuit for measuring the hydraulic pressure therein.
• The separation apparatus further comprises four pairs of a first and secondpinch valve members700,710 that are mounted on the rotor around the opening of thecentral container340. Each pair ofpinch valve members700,710 faces oneseparation cell4000, with which it is associated. Thepinch valve members700,710 are designed for selectively blocking or allowing a flow of liquid through a flexible plastic tube, and selectively sealing and cutting a plastic tube. Eachpinch valve member700,710 comprises an elongated cylindrical body and a head having agroove720 that is defined by a stationary upper jaw and a lower jaw movable between an open and a closed position. Thegroove720 is so dimensioned that one of thetubes400,170,180 of the bag set shown inFIG. 4 can be snuggly engaged therein when the lower jaw is in the open position. The elongated body contains a mechanism for moving the lower jaw and it is connected to a radio frequency generator that supplies the energy necessary for sealing and cutting a plastic tube. Thepinch valve members700,710 are mounted inside thecentral container340, adjacent the interior surface thereof, so that their longitudinal axes are parallel to therotation axis310 and their heads protrude above the rim of thecontainer340. The position of a pair ofpinch valve members700,710 with respect to aseparation bag1000 and thetubes400,170,180 connected thereto when theseparation bag1000 rests in theseparation cell4000 associated with this pair ofpinch valve members700,710 is shown in doted lines inFIG. 4. Electric power is supplied to thepinch valve members700,710 through a slip ring array that is mounted around a lower portion of therotor shaft320.
• The separation apparatus further comprises four pairs ofsensors730,740 (seeFIGS. 6 and 7) for monitoring the separation of the various components occurring within each separation bag when the apparatus operates. Each pair ofsensors730,740 is embedded in thelid470 of thecontainer410 of eachseparation cell4000 along the medianlongitudinal axis420 of thecontainer410, afirst sensor730 being located the farthest and asecond sensor740 being located the closest to therotation axis310. When aseparation bag1000 rests in thecontainer410 and thelid470 is closed, the first sensor730 (later the bag sensor) faces the upper triangular part of theseparation bag1000 and the second sensor740 (later the tube sensor) faces the proximal end of thefirst tube400. Thebag sensor730 is able to detect blood cells in a liquid. Thetube sensor740 is able to detect the presence of absence of liquid in thetube400 as well as to detect blood cells in a liquid. Eachsensor730,740 may comprise a photocell including an infrared LED and a photo-detector. Electric power is supplied to thesensors730,740 through the slip ring array that is mounted around the lower portion of therotor shaft320.
• The separation apparatus further comprises a first balancing means for initially balancing the rotor when the weights of the fourseparation bags1000 contained in theseparation cells4000 are different. The first balancing means substantially comprises the same structural elements as the elements of the component transferring means described above, namely: four expandablehydraulic chambers510 interconnected by a peripheralcircular manifold520, and a hydraulicliquid pumping station6000 for pumping hydraulic liquid into thehydraulic chambers510 through arotor duct560, which is connected to thecircular manifold520. In order to initially balance the rotor, whose fourseparation cells4000 contain four discrete volumes of a composite liquid that may not have the same weight (because the four volumes may be not equal, and/or the density of the liquid may slightly differ from one volume to the other one), thepumping station6000 is controlled so as to pump into the interconnectedhydraulic chambers510, at the onset of a separation process, a predetermined volume of hydraulic liquid that is so selected as to balance the rotor in the most unbalanced situation. For whole blood, the determination of this balancing volume takes into account the maximum difference in volume between two blood donations, and the maximum difference in hematocrit (i.e. in density) between two blood donations. Under centrifugation forces, the hydraulic liquid will distribute unevenly in the fourseparation cells4000 depending on the difference in weight of theseparation bags1000, and balance the rotor. In order to get an optimal initial balancing, the volume of thecavity430 of theseparation cells4000 should be selected so that thecavities430, whatever the volume of theseparation bags1000 contained therein, are not full after the determined amount of hydraulic liquid has been pumped into theinterconnected expansion chambers510.
• The separation apparatus further comprises a second balancing means, for balancing the rotor when the weights of the components transferred into thesatellite bags200,300,150 in thecentral container340 are different. For example, when two blood donations have the same hematocrit and different volumes, the volumes of plasma extracted from each donation are different, and the same is true when two blood donations have the same volume and different hematocrit. As shown inFIGS. 5 and 6 the second balancing means comprises four flexiblerectangular pouches810,820,830,840 that are interconnected by four tube sections (not shown), each tube section connecting two adjacent pouches by the bottom thereof. Thepouches810,820,830,840 contain a volume of balancing liquid having a density close to the density of the composite liquid. The volume of balancing liquid is so selected as to balance the rotor in the most unbalanced situation. The fourpouches810,820,830,840 are so dimensioned as to line the inner surface of thecentral container340 and to have an internal volume that is larger than the volume of balancing liquid so that the balancing liquid can freely expand in any of thepouches810,820,830,840. In operation, if, for example, foursatellite bags200 respectively adjacent to the fourpouches810,820,830,840 receive different volumes of a plasma component, the foursatellite bags200 will press unevenly, under centrifugation forces, against the fourpouches810,820,830,840, which will result in the balancing liquid becoming unevenly distributed in the fourpouches810,820,830,840 and compensating for the difference in weight in thesatellite bags200.
• The separation apparatus further comprises acontroller900 including a control unit (e.g. a microprocessor) and a memory unit for providing the microprocessor with information and programmed instructions relative to various separation protocols (e.g. a protocol for the separation of a plasma component and a blood cell component, or a protocol for the separation of a plasma component, a platelet component, and a red blood cell component) and to the operation of the apparatus in accordance with such separation protocols. In particular, the microprocessor is programmed for receiving information relative to the centrifugation speed(s) at which the rotor is to be rotated during the various stages of a separation process (e.g. stage of component separation, stage of a plasma component expression, stage of suspension of platelets in a plasma fraction, stage of a platelet component expression, etc), and information relative to the various transfer flow rates at which separated components are to be transferred from theseparation bag1000 into thesatellite bags200,300,150. The information relative to the various transfer flow rates can be expressed, for example, as hydraulic liquid flow rates in the hydraulic circuit, or as rotation speeds of thestepper motor640 of thehydraulic pumping station6000. The microprocessor is further programmed for receiving, directly or through the memory, information from thepressure gauge680 and from the four pairs ofphotocells730,740 and for controlling thecentrifuge motor360, thestepper motor640 of thepumping station6000, and the four pairs ofpinch valve members700,710 so as to cause the separation apparatus to operate along a selected separation protocol.
• According to a first separation protocol, four discrete volumes of blood are separated into a plasma component, a first cell component comprising platelets, white blood cells, some red blood cells and a small volume of plasma (later the “buffy coat” component) and a second cell component mainly comprising red blood cells. Each volume of blood is contained in aseparation bag1000 of a bag set represented inFIG. 4, in which it has previously been collected from a donor using thecollection tube500. After the blood collection, thecollection tube500 has been sealed and cut close to the separation bag. Typically, the volumes of blood are not the same in the fourseparation bags1000, and the hematocrit varies from oneseparation bag1000 to another one. Consequently, theseparation bags1000 have slightly different weights.
• The first stage begins by loading the four bag sets into the fourseparation cells4000. Thelids470 are closed and locked, whereby theseparation bags1000 are secured by their upper edge to the containers410 (thepins480 of the securing means pass then through theholes800 in the upper corner of theseparation bags1000 and engage therecesses490 or the securing means).
• Thetubes170 connecting theseparations bags1000 to theplasma component bags200, through theT connectors160, are inserted in thegroove720 of the firstpinch valve members700. Thetubes180 connecting theseparations bags1000 to the buffycoat component bags150, through theT connector160, are inserted in thegroove720 of the secondpinch valve members710. The fourplasma component bags200, the four buffycoat component bags150, the four red bloodcell component bags300 and the fourleukoreduction filters130 are inserted in thecentral compartment340 of the rotor. The fourplasma component bags200 are respectively placed in direct contact with thepouches810 to840 of the second balancing means. Thepinch valve members700,710 are closed and thebreakable stoppers90 in thetubes400 connecting theseparation bags1000 to theT connectors100 are manually broken.
• In the second stage, the rotor is balanced in order to compensate for the difference in weights of the separation bags.
• At the onset of the second stage, all thepinch valve members700,710 are closed. The rotor is set in motion by thecentrifuge motor360 and its rotation speed increases steadily until it rotates at a first centrifugation speed. Thepumping station6000 is actuated so as to pump a predetermined overall volume of hydraulic liquid into the fourhydraulic chambers510, at a constant flow rate. This overall volume of liquid is predetermined taking into account the maximum variation of weight between blood donations, so that, at the end of the second stage, the weights in thevarious separation cells400 are substantially equal and the rotor is substantially balanced, whatever the specific weights of theseparation bags1000 that are loaded in theseparation cells4000. Note that this does not imply that theinternal cavity430 of theseparation cells4000 should be filled up at the end of the balancing stage. For the purpose of balancing the rotor, it suffices that there is enough hydraulic liquid in theseparation cells4000 for equalizing the weights therein, and it does not matter if an empty space remains in each separation cell4000 (the size of this empty space essentially depends on the volume of theinternal cavity430 of aseparation cell4000 and the average volume of a blood donation). Because thehydraulic chambers510 are interconnected, the distribution of the overall volume of hydraulic liquid between theseparations chambers4000 simply results from the rotation of the rotor. When the weights of theseparation bags1000 are the same, the distribution of the hydraulic liquid is even. When they are not, the distribution of the hydraulic liquid is uneven, and the smaller the weight of aspecific separation bag1000, the larger the volume of the hydraulic fluid in the associatedhydraulic chamber510.
• In the third stage, the blood within theseparation bag1000 is sedimented to a desired level.
• At the onset of this stage, all pinchvalve members700,710 are closed. The rotor is rotated at a second centrifugation speed (high sedimentation speed or “hard spin”) for a predetermined period of time that is so selected that, whatever the hematocrit of the blood in theseparation bag1000, the blood sediments in each of theseparation bag1000 at the end of the selected period to a point where the hematocrit of the outer red blood cell layer is about 90 and the inner plasma layer does not substantially contain any more cells, the platelets and the white blood cells forming then an intermediary layer between the red blood cell layer and the plasma layer.
• In the fourth stage a plasma component is transferred into theplasma component bag200.
• At the onset of this stage, the rotation speed is decreased to a third centrifugation speed, the four firstpinch valve members700 controlling access to theplasma component bag200 are opened, and thepumping station6000 is actuated so as to pump hydraulic liquid at a first constant flow rate into thehydraulic chambers510 and consequently squeeze theseparation bag1000 and cause the transfer of plasma into theplasma component bags200.
• When blood cells are detected by thebag sensor730 in theseparation cell4000 in which this detection occurs first, thepumping station6000 is stopped and the corresponding firstpinch valve member700 is closed, either immediately of after a predetermined amount of time selected in view of the volume of plasma that it is desirable in the buffy coat component to be expressed in a next stage.
• Following the closure of the first (first) pinch valve member700 (i.e. the first pinch valve of the group of first pinch valve members700) to close, thepumping station6000 is actuated anew so as to pump hydraulic liquid at a second, lower, flow rate into thehydraulic chambers510 and consequently squeeze the threeseparation bags1000 whose outlet is not closed by the corresponding firstpinch valve members700.
• When blood cells are detected by thebag sensor730 in theseparation cell4000 in which this detection occurs second, thepumping station6000 is stopped and the corresponding firstpinch valve member700 is closed (same timing as for the closing of the first (first) pinch valve member to close).
• Following the closure of the second (first)pinch valve member700 to close, thepumping station6000 is actuated anew so as to pump hydraulic liquid at the second flow rate into thehydraulic chambers510 and consequently squeeze the twoseparation bags1000 whose outlet is not closed by the corresponding firstpinch valve members700.
• When blood cells are detected by thebag sensor730 in theseparation cell4000 in which this detection occurs third, thepumping station6000 is stopped and the corresponding firstpinch valve member700 is closed (same timing as for the closing of the first (first) pinch valve member to close).
• Following the closure of the third (first)pinch valve member700 to close, the pumpingstation600 is actuated anew so as to pump hydraulic liquid at the second flow rate into thehydraulic chambers510 and consequently squeeze theseparation bag1000 whose outlet is not yet closed by the corresponding firstpinch valve member700.
• When blood cells are detected by thebag sensor730 in theseparation cell4000 in which this detection occurs last, thepumping station6000 is stopped and the corresponding firstpinch valve member700 is closed (same timing as for the closing of the first pinch valve member to close).
• In the plasma component transfer process described above, the transfer of the four plasma components starts at the same time, run in part simultaneously and stop independently of each other upon the occurrence of a specific event in each separation bag (detection of blood cells by the bag sensor).
• The fourth stage ends when the four firstpinch valve members700 are closed.
• In the fifth stage a buffy coat component is transferred into the buffycoat component bags150.
• Thecontrol unit900 is programmed to start the fifth stage after the four firstpinch valve members700 are closed, upon receiving information from thelast bag sensor730 to detect blood cells.
• At the onset of this stage, the rotation speed remains the same (third centrifugation speed), a first of the four secondpinch valve members710 controlling access to the buffycoat component bags150 is opened, and thepumping station6000 is actuated so as to pump hydraulic liquid at a third constant flow rate into thehydraulic chambers510 and consequently squeeze theseparation bag1000 in theseparation cell4000 associated with the opened secondpinch valve members710 and cause the transfer of the buffy coat component into the buffycoat component bag200 connected to thisseparation bag1000.
• After a predetermined period of time after blood cells are detected by thetube sensor740 in theseparation cell4000 associated with the opened secondpinch valve member710, thepumping station6000 is stopped and the secondpinch valve member710 is closed.
• After the first (second)pinch valve member710 has closed (i.e. the first pinch valve of the group of second pinch valve members710), a second (second)pinch valve member710 is opened, and a second buffy coat component is transferred into a buffycoat component bag200, in the same way as above.
• The same process is successively carried out to transfer the buffy coat component from the two remainingseparation bags1000 into the buffycoat component bag200 connected thereto.
• In the buffy coat component transfer process described above, the transfers of the four buffy coat components are successive, and the order of succession is predetermined. However, each of the second, third and four transfers starts following the occurrence of a specific event at the end of the previous transfer (detection of blood cells by thetube sensor740 or closing of the second valve member710).
• The fifth stage ends when the four secondpinch valve members710 are closed.
• In the sixth stage the centrifugation process is ended.
• Thecontrol unit900 is programmed to start the sixth stage after the four (second)pinch valve members710 are closed, upon receiving information from thelast tube sensor740 to detect blood cells.
• The rotation speed of the rotor is decreased until the rotor stops, thepumping station6000 is actuated so as to pump the hydraulic liquid from thehydraulic chambers510 at a high flow rate until thehydraulic chambers510 are empty, and the first and secondpinch valve members700,710 are actuated so as to seal and cut thetubes170,180. The red blood cells remain in theseparation bag1000.
• When the fifth stage is completed, the four bag sets are removed from the separation apparatus and each bag set is separately handled manually.
• Thebreakable stopper100 blocking the communication between theseparation bag1000 and thetube600 connected thereto is broken, as well as thebreakable stopper140 blocking the communication between thesecond satellite bag300 and thetube600. The storage solution contained in thesecond satellite bag300 is allowed to flow by gravity through theleukoreduction filter130 and into theseparation bag1000, where it is mixed with the red blood cells so as to lower the viscosity thereof. The content of theseparation bag1000 is then allowed to flow by gravity through thefilter130 and into thesecond satellite bag300. The white blood cells are trapped by thefilter130, so that substantially only red blood cells are collected into thesecond satellite bag300.
• While using any whole blood processing devices like the ones described above, it has been observed that if whole blood is processed on the same day as it was collected, platelets clump or aggregate together and coat the blood separation bag. This leads to a significant reduction in platelet yield and decreased quality of the platelet component. A further observation is that platelet clumping/aggregation does not occur if whole blood is not processed the same day as collection, but is stored and processed the next day after being collected.
• A further observation is that when separating blood into components using an automated whole blood processing device, leukoreduction procedures to remove white blood cells from red blood cells are more efficient in freshly collected blood rather than older blood.
• From these observations, it appears that in procedures to separate blood components from whole blood using an automated whole blood processing device, optimal platelet collection and optimal red blood cell leukoreduction have different requirements.
• The machines described above are used to separate previously collected whole blood into components. Previously collected whole blood could be separated into components either the same day as collection, or the next day after collection. How efficient separation of previously collected whole blood is however, depends upon several factors, such as the type of anticoagulant initially used during whole blood collection, and the starting pH of the whole blood to be separated. As discussed above, separation of platelets from whole blood on the same day the blood is collected is not as efficient as separation of platelets from blood which was collected the previous day. “Same day” blood is defined as blood which has been separated into components on the same day it was collected from a donor. “Next day” blood is defined as blood which has been separated into components on the day after collection.
• In same day blood, platelets are more likely to aggregate and/or stick to the bag during the separation process as compared with next day blood. However, reduction of the amount of white blood cells in red blood cells via leukoreduction is more efficient in same day blood as compared to blood which has been previously collected, cooled and stored. Furthermore, many commercially available leukoreduction filters on the market are indicated for use at room temperature, which is the temperature of same day blood. Filters are more likely to plug during white blood cell filtration with next day cooled blood.
• Same day blood has an average pH of between about 7.1 and 7.2. Next day blood has a pH of between about 6.8 to 6.9 at 37° C.
• To address the seemingly antagonistic requirements for platelet collection and red blood cell leukoreduction, the pH of freshly collected whole blood may be modified to allow for immediate separation into the desired components, especially platelets. This could be done by changing the pH of the anticoagulant used in whole blood collection to make it more acidic. The term acidic pH means that the anticoagulant is buffered sufficiently such that the resultant mixture of whole blood and anticoagulant has a pH of around 6.8.
EXAMPLES
• Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.
Example 1
• As discussed above, CPD is the anticoagulant currently used in whole blood collection. CPD has mean pH of around 7.0. The pH of CPD could be lowered to prevent platelets from clumping and sticking to the bag. An acid such as citric acid could be added to CPD to lower the resulting pH of the blood plus CPD to around 6.8. This would have the same effect as storing the blood overnight, which, as mentioned above, also lowers the pH of the blood. The effect would be immediate however, thereby allowing the freshly collected whole blood to be separated and the red blood cells to be leukoreduced efficiently on the same day as collection, while preventing platelets from clumping and/or sticking to the bag.
• Whole blood and CPD was acidified by adding 60 g isotonic citric acid to 1000 mL of water. 4-5 g of this solution was added to 450 mL whole blood collected in 63 mL of CPD.
• The acidifying solution could be added directly to whole blood and anticoagulant contained in bag2 (seeFIG. 1) or bag1000 (seeFIG. 4), or could be added to the whole blood and anticoagulant in the separation bag5 (seeFIG. 1). The acidifying solution could also be added to the anticoagulant before it is mixed with the collected whole blood.
• In another embodiment, the pH of the anticoagulant could be lowered by adding CO₂to the anticoagulant. The CO₂could be added before the anticoagulant is added to the whole blood, or could be added after the whole blood is mixed with the anticoagulant. The CO₂could be bubbled through the fluid using any commonly available means.
Example 2
• Alternatively, an anticoagulant could be used which has a lower initial pH (is more acidic) then CPD. One such anticoagulant is ACDA, which has a mean pH of around 6.8. ACDA could be substituted for CPD.
• Example 2 shows the platelet collection results obtained from units of whole blood separated on the same day as collection using either of the above described whole blood separation apparatuses. Whole blood units were collected in either ACDA or in CPD. Platelets were counted at 0 time after collection (T=0), 1 hour (T=1 hour) and 24 hours (T=24 hours) after collection. As can be seen from the table, units initially collected in ACDA produced a superior platelet yield (or cell count) and far less clumping (platelet recovery) then units initially collected in CPD.

• 						Platelet
  		Platelet		Platelet	Platelet	Recovery
  	Platelet	Recovery	Platelet	Recovery	Count	(%)
  	Count	(%)	Count	(%)	T = 24	T = 24
  	T = 0 hours	T = 0 hours	T = 1 hour	T = 1 hour	hours	hours

  Same day	8.9 × 10	77%	9.4 × 10	81%	9.9 × 10	88%
  blood
  collected in
  ACDA
  Same day	3.4 × 10	37%	5.9 × 10	64%	6.2 × 10	67%
  blood
  collected in
  CPD

• As can be seen from Examples 1 and 2, by changing the pH of the anticoagulant used, the platelets will clump and/or stick to the bag much less than if anticoagulant having a higher pH is used. It can be extrapolated from this data that leukoreduction will be more efficient because the whole blood is separated into components on the same day it was collected. This principle is shown in Example 3 below.
Example 3
• In Example 3, whole blood was collected in ACDA and separated the next day after overnight cold storage. Separated red blood cells were then leukoreduced (shown as POST-O/N storage in the table below). These results were compared to whole blood collected in ACDA and separated and leukoreduced the same day as collection (shown as PRE-O/N storage). As seen below, leukoreduction of next day blood collected in ACDA was not compromised. Data shown is the average of 6 samples.

• 		Platelet
  		Count per		WBC Count
  		Unit whole		per Unit
  	Platelet Count	blood	WBC Count	whole blood
  	(×103/μL)	(×1010)	(×103/μL)	(×1010)

  PRE-O/N	60	1.38	3.23	0.74
  storage
  WBC
  filtration
  POST-O/N	18.83	0.468	0.55	0.69
  storage
  WBC
  filtration

Example 4
• If next day blood is to be separated and leukoreduced, in another embodiment, a buffering solution to increase the pH of the separated red blood cells may be added to the separated red blood cells before leukoreduction to enable more efficient filtration.
• A solution which could increase the pH of red blood cells separated from next day blood could be added to separation bag5 (seeFIG. 1) or bag1000 (seeFIG. 4). Once the pH of the separated red blood cells reaches a pH of around 6.8, the red blood cells could then be leukoreduced. Examples of pH increasing solutions include buffers such as phosphate buffers which would raise the pH of the solution to between around 6.7 to 7.0.
• In another embodiment, the pH of any solutions commonly used to store red blood cells could be increased to provide a more optimal pH for leukoreduction of red blood cells. A storage solution having an increased pH could have a pH of around 6.7 to 7.0. A storage solution having increased pH could be in bag4 (seeFIG. 1) or bag300 (seeFIG. 4).
• It is understood for the purposes of this disclosure that various changes and modifications may be made to the invention that are will within the scope of the invention. Numerous other changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the methods disclosed herein.

Claims (23)

1. A method of collecting and separating whole blood into at least one component comprising the steps of:

adding an anticoagulant having an acidic pH to a bag for collecting and/or separating whole blood;

collecting whole blood in the bag;

loading the bag containing anticoagulated whole blood on a rotor;

spinning the bag on the rotor to separate the whole blood into at least one component; and

squeezing the bag on the rotor to transfer the at least one component from the separation bag into a satellite bag.

2. The method ofclaim 1 wherein the step of adding an anticoagulant further comprises adding ACDA.

3. The method ofclaim 1 wherein the step of adding an anticoagulant further comprises adding CPD acidified with citric acid.

4. The method ofclaim 1 wherein the step of adding an anticoagulant further comprises adding CPD acidified with carbon dioxide.

5. The method ofclaim 1 wherein the anticoagulated whole blood has a mean pH of between 6.7 and 7.0.

6. The method ofclaim 1 wherein the anticoagulated whole blood has a mean pH of around 6.8.

7. The method ofclaim 1 wherein the step of separating the whole blood into at least one component further comprises separating a red blood cell component.

8. The method ofclaim 7 wherein the separating step further comprises leukoreducing the separated red blood cell component.

9. The method ofclaim 1 wherein the separating step further comprises separating the whole blood on the same day the blood was collected.

10. The method ofclaim 1 wherein the separating step further comprises separating the whole blood on the day after the blood was collected.

11. A method of separating red blood cells from previously collected and stored whole blood comprising the steps of:

collecting whole blood in CPD anticoagulant;

storing the anticoagulated whole blood overnight;

loading the anticoagulated whole blood on a rotor;

spinning the rotor to separate the whole blood into at least a red blood cell component;

squeezing the blood on the rotor to transfer at least the red blood cell component into a satellite bag; and

increasing the pH of the separated red blood cell component in the satellite bag.

12. The method ofclaim 11 further comprising the step of leukoreducing the red blood cell component having increased pH.

13. The method ofclaim 11 wherein the step of increasing the pH of the separated red blood cell component further comprises adding a phosphate buffer.

14. The method ofclaim 11 wherein the step of increasing the pH of the separated red blood cell component further comprises adding a red blood cell storage solution that has an increased pH.

15. A method for separating whole blood into components comprising the steps of:

collecting whole blood into a bag containing an anticoagulant having an acidic pH;

loading the bag containing the anticoagulant and whole blood onto a rotor;

spinning the rotor to separate the whole blood into a plasma component, a platelet component and a red blood cell component;

squeezing the bag on the rotor to transfer the plasma component into a first satellite bag;

squeezing the bag on the rotor to transfer the platelet component into a second satellite bag; and

squeezing the bag on the rotor to transfer the red blood cell component into a third satellite bag.

16. The method ofclaim 15 further including the step of increasing the pH of the red blood cell component.

17. The method ofclaim 16 wherein the step of increasing the pH of the red blood cell component further comprises adding a red blood cell storage solution which has an increased pH to increase the pH of the red blood cells.

18. The method ofclaim 15 further comprising a step of flowing the red blood cell component through a leukoreduction filter.

19. The method ofclaim 15 further comprising a step of storing the collected whole blood and anticoagulant overnight before the separating step.

20. A pre-connected bag and solution set comprising:

a collection bag containing anticoagulant solution having an acidic pH;

a separation bag pre-connected via transfer tubing to the collection bag; and

at least one satellite bag pre-connected via transfer tubing to the separation bag.

21. The pre-connected bag and collection set ofclaim 20 further comprising a plurality of satellite bags pre-connected to the separation bag via transfer tubings.

22. A pre-connected bag and solution set comprising:

a separating bag containing anticoagulant solution having an acidic pH; and

at least one satellite bag pre-connected via tubing to the separating bag.

23. The pre-connected bag and separation set ofclaim 22 further comprising a plurality of satellite bags preconnected via tubings to the separating bag.

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